1. Field of the Invention
This application relates to the field of medical devices and more particularly to the field of thromboresistant coatings for medical devices.
2. Description of Related Art
Arteriosclerosis is a condition that detrimentally affects many individuals. Untreated, arteriosclerosis may lead to severe consequences, including heart damage, heart attack and death. Known treatments for arteriosclerosis have had limited success. Transluminal balloon angioplasty, wherein a balloon is inserted via a catheter into the artery of the patient and expanded, thereby simultaneously expanding the partially closed artery to a more open state, is a well-known treatment for arteriosclerosis, but long-term benefits of balloon angioplasty are limited by the problems of occlusion and restenosis, which result in re-closure of the artery.
A variety of intravascular stents and prostheses have been developed to support diseased arteries and thereby inhibit arterial closure after angioplasty. In particular, expandable intraluminal stents have been developed in which a catheter is used to implant a stent into the artery of the patient in a minimally invasive manner.
Like other foreign bodies placed into arteries, stents can result in coagulation or thrombosis in the intravascular environment. Thrombosis can inhibit blood flow through the stent, diminishing its effectiveness, or can cause clotting, which can threaten the life of the patient. Accordingly, methods of reducing thrombotic activity have been sought to reduce the negative side effects caused by certain stents.
A number of coatings have been developed for medical devices that are intended to promote compatibility between a particular medical device and the environment in which the medical device resides. Some of these coatings, known as thromboresistant coatings, are intended to reduce the thrombosis often associated with insertion of a foreign object, such as a medical device, into the interior of the body.
Heparin, or heparinic acid, arteven, or leparan, is a glycosaminoglycan with well-known anticoagulant activity. Heparin is biosynthesized and stored in mast cells of various animal tissues, particularly the liver, lung and gut. Heparin is known to have antithrombotic activity as a result of its ability to bind and activate antithrombin III, a plasma protein which inhibits several enzymes in the coagulation cascade. It has been hoped that heparin coatings, by inhibiting thrombogenesis, can improve the therapeutic outcomes derived from intra-vascular medical devices, such as stents.
However, known heparin coatings are subject to a number of defects, including incompatibility with the organism and/or microscopic features of the surface to be coated, excessive thickness, difficulty in application, and insufficient durability. For example, several known coatings are based upon simultaneous coulombic interactions between heparin and tri(dodecyl)methylammonium chloride, which is also referred to herein as heparin-TDMAC, and hydrophobic interactions between the quaternary ammonium ion of heparin-TDMAC and the surface of the device. Due to the relative weakness of hydrophobic interactions, such coatings typically leach away from the substrate to which they are applied within a few hours; coatings of this type, therefore, are not generally durable enough to provide beneficial therapeutic results.
Other known coatings comprise silanes having a pendent amino or vinyl functionality. In the fabrication of these coatings, a base layer of silane is applied initially to the surface, followed by the application to the base layer of a second layer comprising antithrombogenic biomolecules, such as heparin. It is necessary that the pendent groups of the base layer of silane be both complementary and accessible to groups on heparin. In some such coatings, a silane with terminal amino functionality is applied to a substrate to form a first layer, followed by application of heparin in solution to form the second layer. In certain examples of this strategy, the amino functionality of the silane base layer reacts with an aldehyde-containing heparin derivative to form a Schiff base and thereby covalently attach the biomolecule to the base layer. In another group of coatings of this general class, a base layer comprising a silane with a vinyl functional group is applied to a surface, followed by covalent attachment, via free radical chemistry, of a heparin-containing derivative to the base layer.
Some of the known coatings have been found lacking in bioeffectiveness and stability. Modifications made in these coatings utilize additional coatings of polymeric matrices comprising reactive functionalities. The multi-step process required to fabricate the polymeric matrices necessary in these approaches increases the thickness of the resulting coatings. Thick coatings present a number of difficulties. First, thick coatings increase the profile of the medical device in the intravascular environment. A stent with a thick profile, for example, can reduce blood flow, thereby undermining the therapeutic benefit of the stent. A thick coating may also render the coating itself more vulnerable to pitting, chipping, cracking, or peeling when the stent is flexed, crimped, expanded, or subjected to intravascular forces. Any of the foregoing results of excessively thick coatings may reduce the antithrombogenic characteristics of the stent. Moreover, the likelihood of pitting is hypothesized to be greater in thick coatings, and pits in a coating may increase the susceptibility to galvanic corrosion of the underlying surface. Because their fabrication requires additional steps, coatings comprising multiple layers may also be more difficult and expensive to manufacture.
Accordingly, a need exists for a thromboresistant coating that is thin, durable, and biocompatible, and that may be applied in a single coating.
Coatings are provided herein in which biopolymers may be covalently linked to a substrate. Such biopolymers include those that impart thromboresistance and/or biocompatibility to the substrate, which may be a medical device. Coatings disclosed herein include those that permit coating of a medical device in a single layer, including coatings that permit applying the single layer without a primer. It should be understood that it may be advantageous in some circumstances to apply double layers of the coatings, such as to cover an area of a medical device that is used to hold the device while a first layer is applied. Thus, single, double and multiple layers of coatings are encompassed by the coatings disclosed herein.
The coatings disclosed herein include those that use an adduct of heparin molecules to provide thromboresistance. The heparin molecules may comprise heparin-tri(dodecyl)methylammonium chloride complex. Uses of the term xe2x80x9cheparinxe2x80x9d herein should be understood to include heparin, as well as any other heparin complex, including heparin-tri(dodecyl)methylammonium chloride complex.
The coatings described herein further include those that use a silane or siloxane to covalently link a biopolymer to a substrate. The coatings include those derived from silanes or siloxanes comprising isocyanate and other electrophilic functionality.
The disclosed coatings include those that can be applied without a base or primer layer.
Coatings are also included that provide a thin and durable coating wherein the thickness of said coating can be controlled by application of single or multiple layers.
Coatings are provided wherein thromboresistance activity can be modified by choice of appropriate amounts of heparin-TDMAC complex and silane/siloxane.
Thin, durable coatings are provided having controllable bioactivity.
Single or multi-layer coatings disclosed herein are designed to impart thromboresistance and/or biocompatibility to a medical device. In one embodiment, the coating provides for covalent linking of heparin to the surface of the medical device.
One coating formulation of the present invention initially consists of heparin-TDMAC complex, organic solvent and silane/siloxane. Wetting agents may be added to this formulation. A silane or siloxane is chosen that has an optional organic chain between the isocyanate (or other functionality) and silicon atom. The isocyanate functionality reacts with an amino or hydroxyl group on the heparin (or other biopolymer) molecule. After the reaction, the formulation contains covalent adducts of heparin and silane, in addition to organic solvent and other additives. Unreacted silane or heparin-TDMAC complex may be present in the formulation, depending on the relative amounts of the reagents utilized.
Once the coating formulation is applied to a device, the silane/siloxane end group of the adduct mentioned above adheres to the substrate surface, and a network, or film, containing heparin-TDMAC complexes is created on the surface of a substrate. Heparin molecules in the heparin-TDMAC complex are known to have anticoagulant properties. When exposed to blood, heparin molecules inactivate certain coagulation factors, thus preventing thrombus formation.
The direct adherence of the silane/siloxane end group to the substrate means that the coating may be applied to a wide range of medical device materials without the use of a base/primer layer. The covalent bond between the surface and the silicon of the silane/siloxane comprising the heparin-TDMAC complex provides superior durability compared to known coatings.
The coating can be applied by dip coating, spray coating, painting, plasma deposition, solvent swelling or wiping. For covered devices, plasma deposition and solvent swelling are preferred modes.
The coating can be thin and durable. The coating thickness can be controlled in a number of ways, e.g., by the application of single or multiple layers. Since the coating process described herein may be a one-step process, coating thickness is not increased as a result of the need to apply multiple layers, as in certain known coating methods.
In one preferred aspect, very thin coatings of suitably derivatized silane or siloxane can be prepared using a plasma deposition process. Accordingly, it is a further aspect of the present invention to provide a medical device having a thromboresistant coating produced by, or produceable by, such a plasma deposition technique.
In another preferred aspect using medical devices covered with a fluoropolymeric material, a silane or a silane-biopolymer is first dissolved in an appropriate organic solvent which, upon contact with the substrate, swells the substrate surface and carries the solute into the surface-modified substrate. Upon drying the solvent, the solute (either the silane or siloxane or the silane-biopolymer) remains embedded into the surface of the substrate thereby providing a thin coating layer with increased resistant to displacement, cracking or leaching. When the silane or siloxane, alone, is embedded into the covering, further contact with a biopolymer creates the covalent thromboresistant complex. Accordingly, it is a further aspect of the present invention to provide a medical device having a thromboresistant coating produced by, or produceable by, a solvent swelling technique.
The bioeffectiveness of the coatings can be controlled by selecting appropriate amounts of reactants. In particular, the thromboresistance activity of the coating can be controlled by modifying the amount of heparin-TDMAC complex in the coating.
Single or multi-layer coatings are provided herein that are designed to impart thromboresistance and/or biocompatibility to a medical device. In an embodiment of the invention, the coating provides for the covalent linking of heparin molecules to a substrate.
A heparin molecule is understood to contain a specific art-recognized pentasaccharide unit that displays antithrombogenic qualities. Covalent linkage of a heparin molecule to a surface is understood to affect at least one, but not all, of the hydroxyl and amino moieties comprised by that molecule; the covalently linked heparin, therefore, presents a thromboresistant surface to the environment surrounding the coated substrate. Different methods and formulations for covalently linking heparin to the surface may affect different sites on the heparin molecules, so that different formulations will provide different levels of anti-thrombogenicity.
One coating formulation of the present invention initially consists of heparin-TDMAC complex, organic solvent and a silane or siloxane. Other biopolymers may be used in place of or in addition to heparin-TDMAC complex, and such biopolymers may be covalently linked to a substrate according to the present invention. Such biopolymers may be those that provide thromboresistance, or those that have other desired bioactivity.
The silane or siloxane provided may have functionality capable of reacting with a nucleophilic group, e.g., a hydroxyl or amino group. In particular, the silane may comprise isocyanate, isothiocyanate, ester, anhydride, acyl halide, alkyl halide, epoxide, or aziridine functionality. Preferably, the silane or siloxane comprises isocyanate functionality.
The silane or siloxane comprising isocyanate functionality may be linked covalently to any biopolymer that provides anti-thrombogenicity. The selected biopolymer may be selected from a group of heparin complexes, including heparin-tridodecylmethylammonium chloride, heparin-benzalkonium chloride, heparin-steralkonium chloride, heparin-poly-N-vinyl-pyrrolidone, heparin-lecithin, heparin-didodecyldimethyl ammonium bromide, heparin-pyridinium chloride, and heparin-synthetic glycolipid complexes. The selected biopolymer may also be another biopolymer that has hydroxyl or amine functional groups that can react with the isocyanate functionality of the silane or siloxane.
The selected biopolymer is preferably capable of dissolving in an organic solvent, as opposed to biopolymers that dissolve only in water. Solubility in organic solvents confers a number of advantages, e.g., elimination of water-mediated decomposition of the isocyanate-containing silane.
In one preferred embodiment, the selected biopolymer is heparin-tri(dodecyl)methylammonium chloride complex.
Wetting agents and other additives may be added to the coatings described herein, to improve the adherence to the substrate, to improve the ease of adding the coatings to a substrate, or for other purposes. A variety of organic solvents may be used, including tetrahydrofuran (THF). Additives may include surface active agents, such as Triton.
The selected silanes or siloxanes may have a linking group, such as an organic chain, between the isocyanate functionality, which covalently links to the heparin molecule, and an end group that is capable of linking to a substrate surface. The end group may link to pendant oxide groups on the substrate surface; in some cases, the pendant oxide groups may be obtained by oxidation of the substrate. Preferred silanes and siloxanes include isocyanatopropyltriethoxysilane, hexamethyldisiloxane, hexamethyldisilane, tetramethyldisiloxane, trimethyldisilane, trimethylsilane, hexamethyldisilizane.
The bioactivity, including thromboresistance, of the disclosed coatings may be selectively modified by controlling the amounts of heparin-tridodecylmethylammonium chloride complex, silane or siloxane comprising isocyanate functionality, and organic solvent, as well as other constituents, to provide the desired thromboresistance. In an embodiment of the coatings, the concentration of the silane or siloxane in the formulation is between about one-half percent and about four percent. In an embodiment, the concentration of heparin-tridodecylmethylammonium chloride in the formulation is between about one-tenth percent and about four percent. One preferred coating is a solution with a formulation of silane or siloxane of about five-tenths percent and a formulation of the heparin-tridodecylmethyl-ammonium chloride complex of about two-tenths percent. In one such preferred solution, the organic solvent is tetrahydrofuran.
Heparin molecules, including those in heparin-TDMAC complex are known to have anticoagulant properties. When exposed to blood, structural elements of heparin molecules inactivate certain coagulation factors, thus preventing thrombus formation.
The coatings described herein may be applied in a single layer. The layer can be formed by reacting silane having isocyanate functionality with a heparin in an organic solvent to form a silane-heparin complex, which can be applied directly to a substrate, such as a metal substrate, in a single-layer coating that can be applied without a primer. The single layer can thus be made sufficiently thin to minimize the problems of peeling, cracking, and other problems that characterize some thicker coatings that require multiple layers, primers, or polymeric matrices for binding to the substrate. Thus, the layers may perform better under the mechanical crimping or expansion of a medical device, such as a stent, to which they are applied, and may perform better in the intravascular environment.
The silane end groups of the monomer that yield the coatings react with oxides or hydroxyl groups on the surface of stainless steel. The stainless steel surface may be oxidized or cleaned and pre-treated, such as with sodium hydroxide, to increase the number of appropriate sites for linking the silane end groups.
For covered medical device surfaces, such as those covered with a polymer like polytetrafluoroethylene (PTFE) or expanded PTFE (TEFLON), fluorinated ethylene propylene, polytetrafluoroethylene-perfluoroalkyl vinyl ether copolymer, polyvinylchloride, polypropylene, polyethylene terephthalate, polyurethane, polyureas, fibrins, broad fluoride, silicone and other biocompatible materials, exposure to organic solvents can swell the surface in a xe2x80x9cspongelikexe2x80x9d manner. A PTFE substrate, or other cellular polymeric substrate, when exposed to a solution of a thromboresistant material, such as heparin-TDMAC, in a suitable organic solvent will swell to contain the solution. Upon drying and/or vacuum removal of the solvent, what is left behind is a substrate surface which contains heparin-TDMAC in an inwardly decreasing concentration gradient of an interpenetrating network.
Suitable solvents include alcohols such as 2-propanol, aromatics such as toluene or xylene, acetone, DMF, ethers such as THF, dimethylacetamide, CH2Cl2, hexane and Freon. It is also contemplated that mixtures or blends of solvents will be preferred based on the chemical and/or physical properties of the thromboresistant coating material or the covered medical device.
Bonding of the silane or siloxane to the covered substrate surface of the medical device can also be accomplished by plasma deposition. Plasma treatment is accomplished using a glow discharge ionization chamber, whereby the substrate is placed in the chamber and the chamber pressure is reduced to a minimal level, preferably below about 0.1 torr, using a vacuum pump. The silane or siloxane is then introduced into the chamber in gaseous form until the chamber pressure reaches a desired level, preferably about 0.3 torr, and then purged again to preferably below about 0.1 torr to reduce contamination from ambient gases. This purge-pump cycle may be repeated as many times as necessary. The final desired pressure of silane or siloxane is typically from about 0.1 torr to about 0.5 torr and most preferably about 0.3 torr. Radio frequency power, preferably from about 10 to about 100 watts, is then generated and applied to the gas in the chamber for a fixed period of time, preferably a period of about 10 to about 20 minutes.
It is necessary to maintain a balance of four factors during the plasma treatment of the substrate surface in order to insure sufficient bonding and to prevent unwanted side reactions at the silane: power (wattage); exposure time (reaction time); gas flow rate; and chamber pressure. Variation of one of these factors may cause adjustment of the other factors in order to achieve the desired result. The wattage should be from about 1 to about 700 watts, preferably less than about 200 watts and most preferably from about 5 to about 60 watts. The flow rate of the gas in the chamber should be about 0.1 to about 500 cubic centimeters per minute (cc/min). The chamber pressure during treatment should be from about 0.1 to about 100 torrs, preferably from about 0.1 to about 10 torrs and most preferably from about 0.1 to about 5 torrs. Exposure time of the substrate surface to the chamber atmosphere varies according to the surface material and is typically from about a few minutes or hours to about 24 hours. Preferably, at 60 watts, 0.1 torr and 0.3 cc/min, a time of about 10 minutes to about 1 hour is sufficient to coat the substrate.
A covered medical device in which a derivatized silane or siloxane has been plasma deposited can then be contacted with a biopolymer, such as heparin-TDMAC, under conditions in which the hydroxyl or amino groups of heparin react with an electrophilic functionality, such as an isocyanate, on the silane or siloxane. The resulting covalent attachment thereby providing the thromboresistant character to the medical device.
The plasma deposition and solvent swelling processes of the present invention are suitable for medical devices covered with a layer of biocompatible material. Preferred biocompatible materials are fluoropolymers, most preferably PTFE and expanded PTFE (TEFLON), fluorinated ethylene propylene, polytetrafluoroethylene-perfluoroalkyl vinyl ether copolymer, polyvinylchloride, polypropylene, polyethylene terephthalate, polyurethane, polyureas, fibrin, broad fluoride, silicone and other biocompatible materials.
Most preferably, medical devices prepared with the plasma deposition and solvent swelling processes of the present invention are xe2x80x9cexpandablexe2x80x9d or xe2x80x9cself-expandingxe2x80x9d stents such as those disclosed in U.S. Pat. No. 5,928,279, which is herein incorporated by reference in its entirety. xe2x80x9cExpandablexe2x80x9d stents are typically formed of spring metal, shape memory alloy (e.g., nickel/titanium alloy), or other material which is resiliently biased toward its fully radially expanded configuration or otherwise capable of expanding to its fully expanded configuration without the need for exertion of extraneous forces. Such stents are initially mounted in a compressed configuration via a delivery catheter, and once delivered, are allowed to return to the expanded configuration in which the stent was initially formed. As used herein, the term xe2x80x9cexpandable stentxe2x80x9d is meant to include self-expanding stents and stents which return to the expanded configuration by the application of temperature. Preferably, the expandable stents to be coated with the plasma deposition or solvent swelling techniques of the present invention are covered with a fluoropolymer.
To improve hydrolytic stability, non-functional silanes can be added to the formulations disclosed herein. Other silanes may be used to link to substrates, such as trihalosilanes, and silanes having methoxy and ethoxy groups. Silanes having triethoxy, trialkoxy, trichloro, and other groups may be provided to yield the covalent linkages present in the coatings disclosed herein. The non-functional silanes may be selected from the group consisting of chain alkyltrialkoxysilanes and phenyltrialkoxysilanes
In an embodiment, the amount of functional silane is preferably selected to provide substantially complete coverage of the substrate surface; that is, it may be desirable to have the single layer cover all of the surface that would otherwise be exposed to the environment in which the substrate will be placed.
The adherence of the silane end group to the substrate means that the coating may be applied to a wide range of medical device materials without the use of base/primer layer. The covalent bond between the heparin-TDMAC complex and the substrate provides a thin and durable coating. The coating""s thickness can be controlled, e.g. by choice of the length of the chain connecting the silane and isocyanate functionalities.
The bioeffectiveness and/or bioactivity of the thromboresistant coating can be controlled by selecting appropriate amounts of reactants. In particular, the thromboresistance activity of the coating can be modified by modifying the amounts of heparin-TDMAC complex and silane in the coating.
Single layers have further advantages in that problems may arise in the extra steps required for the deposition of multiple layers. For example, dust or other particulates may appear between coatings in two-step processes. Also, application of a second layer may tend to reduce reactivity of the first layer in an unpredictable way.
Coatings of the present invention may be applied to medical devices that are placed in the body of a human, or that remain outside the body. Coated medical devices that are placed in the human body may include stents, cathethers, prostheses and other devices. Coated medical devices that remain outside the human body may include tubing for the transport of blood and vessels for the storage of blood. Substrates or medical devices on which the coatings described herein may be applied can include a wide variety of materials, including stainless steel, nitinol, tantalum, glass, ceramics, nickel, titanium, aluminum and other materials suitable for manufacture of a medical device.
The coatings disclosed herein may further include a film-forming agent for the coating. The film-forming agents could slow any leaching of the biopolymer from the coating. The film forming-agent could be added in a second layer, or dissolved simultaneously with the silane and the biopolymer. Appropriate film-forming agents could include cellulose esters, polydialkyl siloxanes, polyurethanes, acrylic polymers or elastomers, as well as biodegradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA, known as PLGA, poly(e-caprolactone), and the like.
To create coatings of the present invention, the silanes and heparin complex are dissolved in a solvent, which may be an organic solvent. The solutions preferably should be substantially anhydrous, because water tends to react with isocyanate groups of the silane molecule. The water may be added after mixing the silane-isocyanate with heparin. In certain embodiments, the silane and heparin are combined in solution, the resulting solution is aged for about one day, the pH is adjusted with a weak acid, and then water is added to hydrolyze silane. The pH of the solution may be adjusted with aqueous acetic acid. Instead of adding water, it is possible to hydrolyze the silane groups by exposure to moist atmospheric conditions. It is desirable to mix the silane and heparin complex in a manner so as to include a slight excess of heparin molecules, so that all of the isocyanate is reacted, preventing adverse reactions between the isocyanate and any water. Moreover, it is desirable to have a single heparin react with each silane isocyanate functional group; this goal is most easily accomplished by starting with an excess of heparin.
Based on experimental results, it was found that, in certain embodiments, solutions of about two-tenths percent heparin complex and about five-tenths percent silane provided effective coatings. However, coatings in a fairly wide range may be effective. Thus, coatings are likely to have some effectiveness in cases in which heparin complex is present in concentrations ranging from about one-tenth of a percent to about twenty percent. Coatings with heparin in concentrations of less than ten percent may be preferable in some formulations. Coatings with heparin in concentrations of less than five percent may be preferable in other formulations. Coatings may be expected to be effective in formulations in which silane is present in a wider range of concentrations as well, including concentrations ranging from about one-tenth of a percent silane to about twenty percent silane.
The thromboresistant characteristics of heparin coatings can be assessed qualitatively and quantitatively, so that methods can be developed that provide uniform coating with a desired amount of bioactivity. Successfully heparinized surfaces give a purple stain when exposed to toluidine blue. After coating, the surface is exposed to a saline solution for a number of days or weeks, and thromboresistance activity is measured as a function of time. Stents and coupons coated as disclosed herein were shown experimentally to display long-lived thromboresistant properties; bioactivity persisted for periods on the order of months, and it will probably endure much longer.
The heparin activity of a sample may be quantified based on its ability to inactivate thrombin. To quantify heparin activity in experimental assays, heparin may be first mixed with human antithrombin III, which binds to create a complex. The heparin-antithrombin III complex can then be mixed with thrombin to produce a ternary complex comprising heparin, thrombin, and antithrombin. The heparin then departs this complex and is free to react again with available antithrombin and thrombin to create additional thrombin-antithrombin complexes. Thus, heparin acts as a catalyst for the antithrombin-mediated deactivation of thrombin. The reaction of the active thrombin still left in the solution with a substrate produces a proportional amount of p-nitro aniline exhibiting color. Thus, an assay may be conducted for a spectrophotometric analysis of color, to determine the amount of thrombin left in solution. The more thrombin left in solution, the lower the bioactivity of the heparin. A low level of thrombin in solution indicates a high degree of catalysis of the thrombin-antithrombin reaction, which indicates a high level of thromboresistance provided by the heparin. A baseline comparison for the assay is the very slow reaction of thrombin-antithrombin in the absence of heparin. The results of the assay can be quantified using spectrophotometry. The assay mimics the reactions that occur in the human bloodstream, where thrombin and anti-thrombin circulate at all times. The reaction between antithrombin and thrombin in the body, which is catalyzed by the heparin of the coatings of the present invention, helps suppress the coagulation that results from thrombogenesis on a medical device.
Various methods of making coatings of the present invention are possible, and examples of such methods and certain resulting coatings are as follows. Such methods and coatings are disclosed by way of example, and are not intended to be limiting, as other examples may be readily envisioned by one of ordinary skill in the art. The following examples include methods of providing coatings of the present invention in a single layer, without the need for a primer layer, as well as methods of controlling the bioactivity of the resulting coating. In some instances, experimental results are provided showing sustained bioactivity for the particular coating.
Coatings can be applied in a wide variety of conventional ways, including painting, spraying, dipping, vapor deposition, epitaxial growth, plasma deposition, solvent swelling and other methods known to those of ordinary skill in the art.
To test coatings disclosed herein, infrared scans were performed to demonstrate changes in the isocyanate functionality, through observation of the isocyanate peak (NCO, 2260 or 2270 cm-1) over time. Isocyanatosilane was formulated with different components, including heparin-tridodecylmethylammonium chloride complex(Heparin-TDMAC complex), tetrahydrofuran (xe2x80x9cTHFxe2x80x9d) and Triton (an optional, surface active agent) in solution to determine whether the intensity of the isocyanate peak changed over time. Table 1 shows the observation of the isocyanate functionality for different solution constitutents:
The observation that the isocyanate peak disappears with time in the solution that includes silane, THF and Heparin-TDMAC complex suggests that a reaction occurs between functional groups on heparin and the isocyanate group of silane.
In embodiments of the present invention, the coating formulation contains the following constituents, which may vary in concentrations in different embodiments: Heparin-TDMAC complex, an organic solvent, such as THF, a silane, such as 3-isocyanatopropyl triethoxysilane (OCNxe2x80x94(CH2)3xe2x80x94Si(OEt)3), and Triton (x-100). In a first embodiment, a solution of these constituents was mixed and allowed to sit in order to permit a reaction to occur. Allowing the solution to sit for one day allowed the reaction to occur, but shorter reaction times may well be effective. Before coating the substrate with the solution, the pH was adjusted. Solutions of the above constituents were adjusted to a pH between 4.5 and 5.5 using a solution of acetic acid and water. After adjusting pH, it is desirable to wait for a period of time, such as fifteen minutes, before applying the coating. Once the coating was applied, it was dried in air and cured in an oven. In particular, coatings of the above constituents were dried in air for about twenty minutes and then cured in an oven at eighty-five degrees Celsius for about one hour.
Coatings, derived from the above-described solutions, on coupons and stents were tested in various ways. First, as a qualitative test, coated coupons and stents were dipped in toluidine blue solution and then were screened for the presence of a purple stain. As mentioned above, the presence of a purple stain in this assay indicates the presence of heparin in the sample being assayed. Additionally, the intensity of the purple stain observed in this assay is proportional to the amount of heparin in the sample. Therefore, a comparison of the intensities of the purple stains produced in this assay by a set of samples allows an assignment of the relative amounts of heparin comprised by the coatings of those samples.
As a quantitative test for heparin activity, a heparin activity assay was conducted according to a conventional thrombin inhibition assay technique. The heparin assay permitted determination of the ability of the heparin coating to deactivate thrombin and thus to provide thromboresistance. The purpose of the protocol was to assay for heparin activity based on thrombin inhibition. A number of different reactions are understood to take place in order to determine heparin activity. In the first reaction:
Heparin+ATIII (excess) - - - [Heparin*ATIII]
Heparin reacts with Human Antithrombin III (xe2x80x9cATIIIxe2x80x9d) to yield a Heparin-Antithrombin III complex. In the second reaction:
[Heparin*ATIII]+Thrombin (excess) - - - [Heparin*ATIII*Thrombin]+Thrombin
the Heparin-Antithrombin complex reacts with Thrombin to yield a Heparin-Antithrombin-Thrombin complex. In the third reaction:
S2238+Thrombin - - - peptide+p-nitroaniline (measured at 405 nm)
the amount of the thrombin was measured. As a result, the size of the p-nitroaniline peak measured at 405 nm is inversely proportional to the amount of heparin present.